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HPLC Analysis of Acetaminophen

Tablets with Waters Alliance and


Agilent Supplies

Application Note
Small Molecule Pharmaceuticals

Authors Abstract
Jignesh Shah, Tiantian Li, and HPLC analysis and method validation for acetaminophen (paracetamol) tablets was
Anil Sharma performed using an Agilent Poroshell 120 column and Waters Alliance system,
Agilent Technologies, Inc. equipped with Agilent performance maintenance kits for pump, autosampler, and a
UV lamp. The analysis was performed following the United States Pharmacopeia
(USP) method [1]. The results showed expected linearity, precision, and sample
recovery values, indicating that Agilent supplies for Alliance systems provide
suitable alternatives to Waters parts.

Introduction
Acetaminophen, also known as paracetamol, is a common pharmaceutical widely
used to relieve pain and reduce fever. The preparation and analysis of
acetaminophen is clearly defined in the United States Pharmacopeia (USP) [1], and
can be conducted on different HPLC systems using columns and sample preparation
products from different manufacturers.

This application note focuses on the use of Agilent performance maintenance kits,
including various frequently exchanged parts such as plungers, seals, injection
needles, solvent filters, and so on. We also assessed Agilent lamps for a Waters
Alliance system to evaluate compatibility. The method to analyze paracetamol
tablets was validated. Detection limits, linearity, accuracy and precision, and sample
recovery were determined. These parameters are important indicators of the
functionality of the system, the column, and sample preparation (filtration) products.
Experimental Preparation of drug tablet assay samples
Two different brands of paracetamol tablets, both labeled as
Chemicals and reagents 500 mg acetaminophen in each tablet, were tested. For each
Acetaminophen USP-level standard and HPLC-grade methanol brand, 20 tablets, equivalent to 10 g acetaminophen, were
from Merck were purchased from Hind Associates dissolved in 1 L of DDS, and then 1 mL of the solution was
(Delhi, India). Paracetamol tablets were purchased from a diluted with DDS to 1 L, corresponding to an acetaminophen
local pharmacy store in Manesar, India. Fresh ultrapure water concentration of 10 µg/mL. The diluted solution was then
was obtained from a Milli-Q Integral system equipped with a filtered by syringe filter. The first 10 mL of filtrate was
0.22 µm membrane point-of-use cartridge. discarded, and the rest of the filtrate was used for HPLC
analysis.
Instrumentation
The HPLC analysis was performed with a Waters Alliance Results and Discussion
Separation Module (model no. 2695) with a Waters UV-VIS
detector (model no. 2487). The system was equipped with an After installing Agilent parts and lamp in the Alliance system,
Agilent deuterium lamp (p/n 8005-0704), and parts from an the system was first tested by different qualification tests,
Agilent performance maintenance kit (p/n 8005-0915), including pump flow accuracy, wavelength accuracy, signal
including plungers, plunger seals, diffuser, face seal, solvent noise and drift, injection precision, carry-over, response
filters, syringe, precolumn filter, check valve cartridges, linearity of a caffeine standard, and gradient composition
PTFE washer, needle wash frit, needle assembly, and injector accuracy. All tests were passed, demonstrating the full
seals, among others. Spectral data were obtained with a functionality of the system.
Waters Acquity photodiode array (PDA) detector.
Acetaminophen standards and drug tablet samples were
Conditions analyzed following the USP method [1], and method validation
Parameter Value was done.
Column: Agilent Poroshell 120 EC-C18, 4.6 × 75 mm, 2.7 µm
(p/n 697975-902) Identification
Mobile phase: 1:3 Methanol:water, isocratic
For the purpose of peak identification, a 10 µg/mL
Sample dissolving
solution/diluent (DDS): 1:3 Methanol:water acetaminophen standard was analyzed and its chromatogram
Flow rate: 1.5 mL/min was overlaid with that of the drug assay sample, in Figure 1.
Column temperature: 25 °C The chromatograms matched perfectly, indicating that the
UV detection: 243 nm major peak at 1.04 minutes was from acetaminophen.
Injection volume: 10 µL
Agilent supplies
Syringe filters: Agilent Captiva Premium syringe filter with
regenerated cellulose membrane, 0.45 µm pore size 10 µg/mL Standard
(p/n 5190-5111)
UV detector intensity (AU)

Drug tablet assay


Vials: Amber, write-on spot, 100/pk (p/n 5182-0716)
Vial caps: Blue, screw cap, 100/pk (p/n 5182-0717)

Preparation of standards
USP-level acetaminophen standards were accurately
weighted and dissolved in sample dissolving solution (DDS) to
obtain a stock solution of 20 µg/mL. Proper standards for 0
method development and validation were prepared by 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 5.0
subsequent dilution of the stock solution in DDS. Time (min)

Figure 1. Overlay of 10 µg/mL acetaminophen standard and


drug tablet sample.

2
To assess the purity of the peak, UV absorption spectra of the ×10 –5
1.043
standard and the drug assay were overlapped in Figure 2. 12 A
Both spectra were identical, demonstrating that the peak was 10
LOD
pure.
8

245.6 6

AU
4
Acetaminophen standard
Drug tablet assay 2

0
Absorbance

-2

-4
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Time (min)
×10 –5 1.045
35 B
LOQ
220 240 260 280 300 320 340 360 380 400 30
Wavelength
25
Figure 2. UV absorption spectra of acetaminophen standard
and drug tablet sample. 20
AU

15

Limit of detection (LOD) and 10


limit of quantitation (LOQ) 5
The analyte concentration with a signal-to-noise ratio (S/N) 0
above three was considered as LOD, and analyte
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
concentration with S/N above 10 was considered as LOQ. Time (min)
The LOD and LOQ of acetaminophen were 10 ng/mL and
Figure 3. Chromatograms of LOD and LOQ of acetaminophen.
50 ng/mL, respectively (Figure 3).

Calibration curve linearity 600,000

The method gave a linear response to acetaminophen from 500,000


0.05 to 20 µg/mL. Six calibration standards with
400,000
concentrations of 0.05, 1, 5, 7.5, 15, and 20 µg/mL were y = 26811x – 1313.8
R² = 0.9999
analyzed, and their peak areas were plotted against 300,000
concentration, in Figure 4. The regression coefficient R2 was 200,000
0.9999, indicating good linearity.
100,000

0
0 5 10 15 20 25

Figure 4. Linear response of peak area against concentration


of acetaminophen.

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Accuracy and precision Analysis of marketed drug tablets
Acetaminophen QC standards at 0.5 µg/mL and 15 µg/mL Paracetamol tablets of two different brands, both labeled as
were analyzed and calculated using the previously generated 500 mg/tablet, were dissolved, filtered through an Agilent
calibration curve to demonstrate method accuracy. To assess Captiva syringe filter, and analyzed. Regenerated cellulose
method precision, both QC standards were prepared three was chosen as the filtration membrane since it showed
times, independently (three batches), and each batch was excellent sample recovery for acetaminophen in a previous
injected in a separate sequence with six replicates. The study [2].
intra-batch precision was assessed by the standard deviation
between the replicates in the same sequence, and the Sample recoveries were 97.8% and 100.7%, both of which
inter-batch precision was evaluated by the variation between were well within the USP-specified range of 90 to 110%,
different batches. The results of accuracy and precision showing the excellent filtration recovery of the syringe filter
studies are summarized in Table 1. and reliability of the analysis method. RSDs between three
different sample batches were 0.31% and 0.17%, indicating
The accuracy for both concentrations and all batches was appropriate quantitation.
well within a low error range, below ±2%. The intra-batch and
inter-batch RSD values were all below 0.7%, indicating good
precision.

Table 1. Accuracy and intra-batch and inter-batch precision of


acetaminophen analysis.
QC sample Calculated Intra-batch Inter-batch
concentration Batch concentration RSD (%) RSD (%)
(µg/mL) no. (µg/mL) Accuracy n=6 n=3
0.5 1 0.51 101.08 0.23 0.58
2 0.51 101.41 0.42
3 0.50 100.24 0.19
15 1 15.06 100.41 0.15 0.62
2 14.85 98.98 0.11
3 14.93 99.52 0.07

Table 2. Accuracy and precision of paracetamol drug tablet assays.


Labeled amount Calculated Accuracy RSD
Paracetamol per tablet (mg) amount (mg) (%) (%)
Brand A 500 488.8 97.8 0.31
Brand B 500 503.3 100.7 0.17

4
Conclusions References
Acetaminophen was analyzed according to the USP method, 1. U.S. Pharmacopeia for Acetaminophen Tablets. United
and specificity, linear range, accuracy, precision, and recovery States Pharmacopoeial Convention, Rockfield, MD, USA.
studies were done to validate the method. A Waters Alliance http://www.pharmacopeia.cn/v29240/usp29nf24s0_m20
system was used for HPLC analysis, equipped with 0.html
comprehensive pump and autosampler parts contained in an
2. L. Zhao; W. Long. Syringe Filter Suitability for Sample
Agilent performance maintenance kit, as well as an Agilent
Preparation in Drug Assays; Application Note, Agilent
deuterium lamp for Alliance systems. Expected results were
Technologies, Inc. Publication number 5991-2409EN,
achieved, and no negative impact by using Agilent supplies
2013.
could be identified compared to OEM parts, indicating that
the Agilent supplies functioned as well as the OEM parts.
This work is an example of how the Agilent product portfolio, For More Information
including instrument parts, lamps, columns, and sample
These data represent typical results. For more information on
preparation products, can provide linear, accurate, precise,
our products and services, visit our Web site at
and reliable analysis of pharmaceuticals with Waters Alliance
www.agilent.com/chem.
systems.

5
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Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change


without notice.

© Agilent Technologies, Inc., 2015


Printed in the USA
November 5, 2015
5991-6017EN

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